Melatonin, a well-known antioxidant, has been shown to possess anti-invasive properties for glioma. However, little is known about the effect of melatonin on glioma cell migration and invasion under hypoxia, which is a crucial microenvironment for tumor progress. In addition, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration and invasion. Therefore, we investigated the possible role of these kinases and its related signaling in the regulation of human U251 glioma cells behavior by melatonin under hypoxia. The abilities of migration and invasion of U251 glioma cells were determined by wound healing and transwell assay in vitro. The intracellular production of reactive oxygen species (ROS) was measured by using the fluorescent probe 6-carboxy-2′, 7′-dichorodihydrofluorescein diacetate (DCFH-DA). Immunofluorescence experiments and western blotting analysis were used to detect the expression level of protein. Small interfering RNAs (siRNA) was used to silence specific gene expression. The pharmacologic concentration (1 mM) of melatonin significantly inhibited the migration and invasion of human U251 glioma cells under hypoxia. The inhibitory effect of melatonin was accompanied with the reduced phosphorylation of FAK and Pyk2, and decreased expression of alpha v beta 3 (αvβ3) integrin. Additionally, inhibition of αvβ3 integrin by siRNA reduced the phosphorylation of FAK/Pyk2 and demonstrated the similar anti-tumor effects as melatonin, suggesting the involvement of αvβ3 integrin- FAK/Pyk2 pathway in the anti-migratory and anti-invasive effect of melatonin. It was also found that melatonin treatment decreased the ROS levels in U251 glioma cells cultured under hypoxia. ROS inhibitor apocynin not only inhibited αvβ3 integrin expression and the phosphorylation levels of FAK and Pyk2, but also suppressed the migratory and invasive capacity of U251 glioma cells under hypoxia. These results suggest that melatonin exerts anti-migratory and anti-invasive effects on glioma cells in response to hypoxia via ROS-αvβ3 integrin-FAK/Pyk2 signaling pathways. This provides evidence that melatonin may be a potential therapeutic molecule targeting the hypoxic microenvironment of glioma.

Aims: High immune cell infiltration in gliomas establishes an immunosuppressive tumor microenvironment, which in turn promotes resistance to immunotherapy. Hence, it is important to identify novel targets associated with high immune cell infiltration in gliomas. Our previous study showed that serum levels of beta‐2 microglobulin (B2M) in lower‐grade glioma patients were lower than those in glioblastoma patients. In the present study, we focused on exploring the roles of B2M in glioma immune infiltration.Methods: A large cohort of patients with gliomas from the TCGA, CGGA, and Gravendeel databases was included to explore differential expression patterns and potential roles of B2M in gliomas. A total of 103 glioma tissue samples were collected to determine the distributions of B2M protein levels by immunofluorescent assays. Kaplan‐Meier survival analysis and meta‐analysis were used for survival analysis. GO(Gene‐ontology) enrichment analysis, co‐expression analysis, KEGG(Kyoto Encyclopedia of Genes and Genomes) pathway analysis, and immune infiltration analysis were performed to explore roles and related mechanisms of B2M in glioma.Results: We found that both B2M mRNA and protein levels were abnormally upregulated in glioma samples compared with those from normal brain tissue. B2M expression was correlated with tumor grade and was downregulated in IDH1 mutant samples. Furthermore, B2M was a moderately sensitive indicator for predicting the mesenchymal molecular subtype of gliomas. Interestingly, glioma patients with lower B2M expression had remarkably longer survival times than those with higher B2M expression. Moreover, meta‐analysis showed that B2M was an independent predictive marker in glioma patients. The results of GO enrichment analysis revealed that B2M contributed to immune cell infiltration in glioma patients. In addition, results of KEGG pathway analysis and co‐expression analysis suggested that B2M may mediate glioma immune infiltration via chemokines.Conclusions: We conclude that B2M levels are critical for the survival times of glioma patients, at least in part due to mediating high immune infiltration.

High immune-cell infiltration in glioblastomas (GBMs) leads to immunotherapy resistance. Emerging evidence has shown that zinc finger Asp-His-His-Cyc-type (ZDHHC) palmitoyl transferases participate in regulating tumor progression and the immune microenvironment. In the present study, a large cohort of patients with gliomas from The Cancer Genome Atlas (TCGA) and Rembrandt databases was included to perform omics analysis of ZDHHCs in gliomas. CCK-8, flow cytometry, quantitative real-time PCR, western blotting, and transwell assays were performed to determine the effects of ZDHHC inhibition on glioma cells and microglia. We found that five (ZDHHC11, ZDHHC12, ZDHHC15, ZDHHC22, and ZDHHC23) out of 23 ZDHHCs were aberrantly expressed in gliomas and might play their roles through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. Further results indicated that inhibition of ZDHHCs with 2-bromopalmitate (2-BP) suppressed glioma-cell viability and autophagy, as well as promoted apoptosis. Targeting ZDHHCs also promoted the sensitivity of glioma cells to temozolomide (TMZ) chemotherapy. In addition, the inhibition of ZDHHCs weakened the migratory ability of microglia induced by glioma cells in vitro and in vivo. Taken together, our findings suggest that the inhibition of ZDHHCs suppresses glioma-cell viability and microglial infiltration. Targeting ZDHHCs may be promising for glioma treatments.

Astrocytes are crucially involved in neuroinflammation. Activated astrocytes exhibit at least two phenotypes, A1 (neurotoxic) and A2 (neuroprotective). The A1 phenotype is the major reactive astrocyte phenotype involved in aging and neurodegenerative diseases. Telmisartan, which is an antihypertensive agent, is a promising neuroprotective agent. This study aimed to investigate the effects of telmisartan on the phenotype of reactive astrocytes. Astrocytes were activated by culturing with the conditioned medium derived from lipopolysaccharide-stimulated microglia. This conditioned medium induced early, transient A2 astrocyte conversion (within 24 h) and late, sustained A1 conversion (beginning at 24 h and lasting up to 7 days), with a concomitant increase in the production of pro-inflammatory cytokines (interleukin [IL]-1β, tumor necrosis factor [TNF]α, and IL-6) and phosphorylation of nuclear factor-κB (NF-κB)/p65. Telmisartan treatment promoted and inhibited A2 and A1 conversion, respectively. Telmisartan reduced total and phosphorylated p65 protein levels. Losartan, a specific angiotensin II type-1 receptor (AT1R) blocker, did not influence the reactive state of astrocytes. Additionally, AT1R activation by angiotensin II did not induce the expression of pro-inflammatory cytokines and A1/A2 markers, indicating that the AT1R signaling pathway is not involved in the astrocyte-mediated inflammatory response. A peroxisome proliferator-activated receptor γ (PPARγ) antagonist reversed the effects of telmisartan. Moreover, telmisartan-induced p65 downregulation was reversed by the proteasome inhibitor MG132. These results indicate that telmisartan suppresses activated microglia-induced neurotoxic A1 astrocyte conversion through p65 degradation. Our findings contribute towards the elucidation of the anti-inflammatory activity of telmisartan in brain disorders.